title: [Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].
abstract: OBJECTIVE
  To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.
  
  
  METHODS
  Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.
  
  
  RESULTS
  Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].
  
  
  CONCLUSIONS
  Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).
authors.affiliation: Department of Microbiology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. wenym2010@163.com
authors.forename: Yangming
authors.initials: Y
authors.lastname: Wen
authors.forename: Kaijian
authors.initials: K
authors.lastname: Lan
authors.forename: Junjie
authors.initials: J
authors.lastname: Wang
authors.forename: Jingyi
authors.initials: J
authors.lastname: Yu
authors.forename: Yarong
authors.initials: Y
authors.lastname: Qu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.forename: Fuchun
authors.initials: F
authors.lastname: Zhang
authors.forename: Wanlong
authors.initials: W
authors.lastname: Tan
authors.forename: Hong
authors.initials: H
authors.lastname: Cao
authors.forename: Chen
authors.initials: C
authors.lastname: Zhou
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D016466:CHO Cells; D061505:Cell Surface Display Techniques; D006224:Cricetinae; D003412:Cricetulus; D003716:Dengue Virus / Q000276:immunology*; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D014162:Transfection
chemical_list: D000914:Antibodies, Viral; D007143:Immunoglobulin Heavy Chains